Protein-Based Stable Isotope Probing (Protein-SIP): Applications for Studying Aromatic Hydrocarbon Degradation in Microbial Communities
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Protein-based stable isotope probing (protein-SIP) was developed to link microbial-specific metabolic function to phylogenetic information. The principle of the SIP-concept is the supplement of stable isotope-labelled compounds resulting in the labelling of microorganisms that are capable of utilizing these substrates as carbon source. The sum of all proteins reflects the functional status of the living organisms, so that the degree of heavy isotope incorporation represents a proxy for substrate assimilation and their activity. The main focus of this chapter is on the application of protein-SIP to elucidate metabolic processes in general and in particular those involved in the anaerobic degradation of aromatic hydrocarbons. Thus, the application of protein-SIP is a useful method to investigate the composition and the functional state of microbial communities.
Meta-omics approaches allow a deep insight into the structure and function of microbial communities (Herbst et al. 2015). Metaproteomics also referred to as community proteomics or environmental proteomics has become over the last years metaproteomics, also referred to as community proteomics or environmental proteomics, has become an important tool in studying microbial ecology approaches (von Bergen et al. 2013) allowing to elucidate functional relationships between microbial community members (Bastida et al. 2015). However, metaproteome analysis alone cannot elucidate which protein and their corresponding species is currently active under specific conditions. For this reason, SIP techniques are required to solve this problem (Jehmlich et al. 2008a). During protein-SIP analysis, the metabolism of the labelled substrate can be detected with high precision on the peptide level. The identification of uniquely labelled peptides principally allows the assignment to phylotypes that express the encoding genes of the proteins providing a direct link of metabolic function to single members of the microbial community (Jehmlich et al. 2016). For these reasons, protein-SIP has developed into an important and widely used tool in microbial ecology. Since its development in 2008 (Jehmlich et al. 2008b), the spectrum of used stable isotopes in protein-SIP analyses has continuously broadened. By applying mass spectrometry (MS), the MS spectra provide biologically valuable information: (i) the presence/abundance of peptides/proteins, (ii) the primary amino acid sequence thereby revealing the proposed function, and (iii) the metabolic activity of the corresponding organism as a measure of incorporation of isotopes. Bioinformatics solutions for automatic data evaluation have been developed, and the range of application has been enlarged from microcosms over mesocosms up to in situ environmental sampling. This book chapter focuses on existing studies on protein-SIP and discusses potential trends of its application in environmental microbiology (e.g., labelled water) with a special focus on anaerobic degradation of aromatic hydrocarbons (Grob et al. 2015). Most technical aspects of protein-SIP applications have been reviewed previously (Taubert et al. 2011; von Bergen et al. 2013).
In the coming years, the development in the fields of mass spectrometry and bioinformatics will increase the resolution and specificity of protein-SIP applications. Further integration of comprehensive protein extraction procedures will promote the analysis of samples with lower amounts of protein, which opens the way to investigate natural microbial communities in more detail.
1.1 Protein-SIP Concept
Metabolic incorporation of heavy stable isotopes (usually 13C or 15N) into proteins has become a powerful technique for qualitative and quantitative proteome studies because the assessment of incorporation is a valuable parameter to investigate the general metabolic activity as well as protein turnover rates. In a time course experiment, the accurate quantification of incorporation in protein-SIP enables also the identification of food webs in microbial consortia.
There are two ways of metabolic protein labelling: (i) the use of substrates or nutrients such as [13C7]-glucose or 15N-ammonium which are biochemically incorporated into amino acids and (ii) the use of specifically labelled amino acids that are added to the medium (SILAC, for details see (Chen et al. 2015)). Stable isotope-labelled substrates (D, 13C, 15N, 18O, 34/36S, or nutrients) have become attractive since the mass shift measured by mass spectrometry clearly allows the quantitative assessment of isotope incorporation, and this is an indirect measurement of metabolic activity in respect to a specific substrate or nutrient. Two important parameters can be retrieved from the mass spectrometry data. First, the relative isotope abundance (RIA) describes the number of labelled atoms in a peptide and gives information about the proportion of labelled substrate that was assimilated. The second parameter named labelling ratio (LR) describes the ratio of labelled to natural peptide and refers to protein turnover rates after addition of the labelled substrate. The greatest advantages of protein-SIP are the accurate and very sensitive quantification of incorporation and the physiological information that is obtained concomitantly.
1.2 Calculation of Stable Isotope Incorporation into Peptides
1.3 Protein-SIP Applications
There are several studies published that use protein-SIP in microbial ecology approaches. Exemplarily, we reviewed three applications that benchmark the field of protein-SIP, (i) a time-resolved protein-SIP, (ii) in situ protein-SIP studies, and (iii) labelled water to track general microbial activity.
A sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer was investigated using time-resolved protein-SIP (Taubert et al. 2012). Benzene is a major contaminant in this aquifer, and the initial mechanisms behind its biodegradation under strictly anoxic conditions were not yet entirely clear. The carbon fluxes within the microbial community were investigated by addition of either 13C-benzene or 13C-carbonate. The utilization of the initial carbon source and the metabolic intermediates was relatively quantified, and the functional groups were affiliated to Clostridiales, Deltaproteobacteria, and Bacteroidetes/Chlorobi. Taubert et al. further observed that the Clostridiales-related organisms were involved in benzene degradation putatively by fermentation. This study revealed that protein-SIP can be applied in a time-dependent manner to obtain temporal and taxonomic information within a microbial community.
Polycyclic aromatic hydrocarbon (PAH) degrading bacteria often rely on laboratory enrichments and isolations. Herbst et al. performed the first in situ microcosms with 13C-naphthalene (BACTRAP(R)s) in which the BACTRAPs were exposed to a PAH-contaminated aquifer (Herbst et al. 2013). Briefly, BACTRAPs are in situ microcosms that consist of perforated Teflon tubes of 5 cm length and 1 cm diameter with a perforation of 1 mm which are filled with 1 g of preheated (4 h at 300 °C) activated carbon pellets (Biocoal, Silcarbon Aktivkohle GmbH, Kirchhundem, Germany). Following sterilization and hydration of the BACTRAPs by autoclaving at 121 °C, the BACTRAPs were each loaded with 25 mg [13C6]-naphthalene by dropping the compounds diluted in 1 mL n-hexane on the activated carbon pellets. As main result of this experiment, Burkholderiales, Actinomycetales, and Rhizobiales were the most active microorganisms in the groundwater communities; and the naphthalene degradation pathway showed high 13C incorporation (about 50 atom%). This study convincingly demonstrated that a combination of in situ microcosms with protein-SIP is a suitable tool for the identification of metabolic key players as well as degradation pathways.
In another approach, the microbial processes in constructed wetlands (CWs) under controlled conditions were investigated. CWs are well-established treatment systems for the bioremediation of contaminated waste- and groundwaters (Schroder et al. 2007). While the systems provide high removal efficiencies for numerous organic contaminants, the microbial processes are not yet well known. Plant roots stimulate the microbial degradation activity within the CW by the exudation of organic compounds as well as oxygen (Lagos et al. 2015). Detailed knowledge about the microbial key players and degradation pathways can aid the design of CWs for improved and stable performances. A planted fixed bed reactor (PFR) was designed and operated in a greenhouse (Kappelmeyer et al. 2002). Toluene was added to the inflow to investigate the respective degradation processes. The microbial community composition was assessed by 16S rRNA gene sequencing and protein-SIP by pulsed addition of 13C-labelled toluene and revealed fast degradation during 40 h inside the PFR (Lünsmann et al. 2015). After 20 h the 13C label was detected in bacterial proteins; almost all labelled proteins could be assigned to the order of Burkholderiales, which constituted to only about 20% of the microbial community (Lünsmann et al. 2015). Among them, two bacterial families showed different proportions of 13C-incorporation (RIA) in their proteins, leading to the conclusion that Burkholderiaceae derived more biomass from toluene (73% RIA), than the Comamonadaceae (64% RIA) (Lünsmann et al. 2015). The complete pathway of toluene degradation was retrieved by protein-SIP which demonstrated that toluene degradation was initiated by a monooxygenase yielding p-cresol, followed by a phenol hydroxylase introducing a second hydroxyl functionality group. It became evident that toluene-derived carbon might be fully oxidized via the citric acid cycle or could be stored by the microbes anabolically as polyhydroxyalkanoate (PHA) granules (Lünsmann et al. 2015, 2016). Toluene degradation is not limited by oxygen availability despite the applied concentrations in the PFR, and Burkholderiales may serve as indicators for effective hydrocarbon removal at low oxygen concentrations.
Micro-pollutants [herbicides, pesticides, and pharmaceuticals present in the environment at very low concentrations (ng-μg/L)] have emerged as an important topic in the field of environmental microbiology (Luo et al. 2014). The most important consequence of the low concentration is that these compounds are often co-metabolized (Fischer and Majewsky 2014; Kjeldal et al. 2016). By designing a protein-SIP experiment, the used concentration has to be remarkably exceeding the environmentally relevant concentration to obtain adequate stable isotope incorporation. If the compounds are only co-metabolically transformed, the incorporation into the biomass of the microorganisms will not be enough, and a protein-SIP experiment using the labelled micro-pollutant is not suitable. Still, using labelled water as substrate-independent compound may be a promising approach.
Hydrogen has the highest abundance in proteins, but its application was limited due to a fast H/D-exchange, which will occur in living cells and during sample preparation. In addition, deuterium at high concentrations inhibits enzymes due to kinetic isotope effects. Furthermore, the chromatographic properties of deuterated compounds that originate from the higher hydrophilicity of CD-bonds compared to CH-bonds (Boersema et al. 2009), cause significant changes in the retention time (Zhang et al. 2002). However, the usage of heavy water, labelled with either deuterium or 18-oxygen, has advances and is promising as it assesses the global activity regardless of the used substrate. Some applications successfully demonstrated using D2O in acid mine drainage biofilms (Justice et al. 2014) and soil communities using H218O in DNA (Blazewicz and Schwartz 2011; Schwartz 2009; Schwartz 2007) and RNA (Angel and Conrad 2013; Rettedal and Brozel 2015). Protein-SIP was applied to track the usage of 15NH4 or deuterium oxide for different members of the community (Justice et al. 2014). There were relatively few 15N-enriched archaeal proteins, and all showed low atom percent enrichment consistent with Archaea synthesizing protein using the predominantly 14N biomass derived from recycled biomolecules (Justice et al. 2014). Deuterium oxide was used to detect general microbial activity in the samples. Interestingly, bacterial species showed only little protein synthesis using deuterium oxide which reflects that the exclusive ability of Archaea to synthesize proteins using 2H2O perhaps due to archaeal heterotrophy (Justice et al. 2014). In a combined Raman microspectroscopy, metaproteomics, and protein-SIP approach, the complex metabolic response using D2O labelling allowed the monitoring of metabolic activity combined with a functional characterization of active populations isolates from groundwater (Taubert et al. 2018). In particular, 18-oxygen seems more promising than deuterium as the abiotic HD exchange of acidic hydrogens could cause a dilution of the label once the proteins are in contact with unlabelled water (Englander and Kallenbach 1983).
Metaproteomics of environmental samples is generally limited by three factors: first, the extraction of proteomes (with various biomass availabilities) from samples with background substances; second, the low genetic coverage in protein-coding databases; and third, the high complexity of the microbial community. For the last factor, protein-SIP contributes to an increase of the coverage because it points directly toward the detection of the active members within a community in respect to a specific substrate. Since the introduction of protein-SIP in 2008, all isotopes present in proteins have been used for studies. Thereby, a broad variety of studies based on energy flux (carbon), nitrogen utilization (nitrogen), or markers of general metabolic activity (deuterium and sulfur) have been performed. The latter examples underline the unique potential of protein-SIP for analyzing microbial communities in situ in soil, sediments, aquifers, water bodies, and higher organisms. Still, one of the urgent needs is the development of new de novo approaches where the natural peptide information is not necessary. The identification of the amino acid composition from metabolically labelled isotopes in the MS spectra is generally performed by matching theoretical MS spectra to protein-coding sequence databases. Bioinformatics algorithms rely on monoisotopic precursor mass (natural abundance) for identification; likewise, MetaProSIP relies on the natural abundance precursor for identification and calculation of the dynamic mass shift starting from that.
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